Urease catalysis and structure: X. Alternate bonding-site isozymes of jackbean urease

Three previously uncharacterized, nongenetic urease isozymes have been analyzed by sucrose density gradient sedimentation, gel electrophoresis, and chemical reactivity. The full complement of isozymes could be reliably generated by choosing appropriate levels of NaCl, pH, and ethylene glycol, and was stable for several days in dilute solution. The three forms of interest were found to be quaternary isomers of other isozymes, but differed from them qualitatively in their bonding sites, with disulfide bonds being substituted for noncovalent bonds. The separation of these isomer-pairs during sedimentation and electrophoresis cannot be readily explained by differences in size or charge, but must rather arise from a difference in shape. A simple two-dimensional model can provide the appropriate molecular architecture to satisfy these requirements: Only one of the two half-units in each α-urease molecule undergoes disulfide bonding during polymerization, and it does so with two adjacent molecules, thus producing asymmetric polymers from symmetric starting components.     

The Golgi apparatus and cell polarity: Roles of the cytoskeleton,the Golgi matrix,and Golgi membranes

Membrane trafficking plays a crucial role in cell polarity by directing lipids and proteins to specific subcellular locations in the cell and sustaining a polarized state. The Golgi apparatus, the master organizer of membrane trafficking, can be subdivided into three layers that play different mechanical roles: a cytoskeletal layer, the so-called Golgi matrix, and the Golgi membranes. First, the outer regions of the Golgi apparatus interact with cytoskeletal elements, mainly actin and microtubules, which shape, position, and orient the organelle. Closer to the Golgi membranes, a matrix of long coiled–coiled proteins not only selectively captures transport intermediates but also participates in signaling events during polarization of membrane trafficking. Finally, the Golgi membranes themselves serve as active signaling platforms during cell polarity events. We review here the recent findings that link the Golgi apparatus to cell polarity, focusing on the roles of the cytoskeleton, the Golgi matrix, and the Golgi membranes.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2006

Adult mosquito surveillance was conducted during 2006 at 22 US Army and Air Force installations and two training sites located in six provinces in the Republic of Korea. Adult mosquitoes were collected in New Jersey light traps from 1 May through 15 October 2006 to determine threshold levels to initiate pesticide applications based on trap indices. In total 16 217 adult mosquitoes [13 612 (83.9%) females and 2605 (16.1%) males] comprising 16 species (including five members of the Anopheles sinensis Group) belonging to eight genera were collected. Females of the most commonly collected species were members of the Anopheles sinensis Wiedemann Group (58.9%), followed by Aedes vexans nipponii (Theobald) (21.3%), Culex tritaeniorhynchus Giles (11.8%) and Culex pipiens Coquillett (5.2%). Trap indices varied widely for species over their range, due in part, to their geographical distribution and degree of association with urban or rural communities.     

Accuracy of gastrocnemius muscles forces in walking and running goats predicted by one-element and two-element Hill-type models

Hill-type models are commonly used to estimate muscle forces during human and animal movement—yet the accuracy of the forces estimated during walking, running, and other tasks remains largely unknown. Further, most Hill-type models assume a single contractile element, despite evidence that faster and slower motor units, which have different activation–deactivation dynamics, may be independently or collectively excited. This study evaluated a novel, two-element Hill-type model with “differential” activation of fast and slow contractile elements. Model performance was assessed using a comprehensive data set (including measures of EMG intensity, fascicle length, and tendon force) collected from the gastrocnemius muscles of goats during locomotor experiments. Muscle forces predicted by the new two-element model were compared to the forces estimated using traditional one-element models and to the forces measured in vivo using tendon buckle transducers. Overall, the two-element model resulted in the best predictions of in vivo gastrocnemius force. The coefficient of determination, r², was up to 26.9% higher and the root mean square error, RMSE, was up to 37.4% lower for the two-element model than for the one-element models tested. All models captured salient features of the measured muscle force during walking, trotting, and galloping (r²=0.26–0.51), and all exhibited some errors (RMSE=9.63–32.2% of the maximum in vivo force). These comparisons provide important insight into the accuracy of Hill-type models. The results also show that incorporation of fast and slow contractile elements within muscle models can improve estimates of time-varying, whole muscle force during locomotor tasks.     

Mechanotransduction down to individual actin filaments

The actin cytoskeleton plays an essential role in a cell's ability to generate and sense forces, both internally and in interaction with the outside world. The transduction of mechanical cues into biochemical reactions in cells, in particular, is a multi-scale process which requires a variety of approaches to be understood. This review focuses on understanding how mechanical stress applied to an actin filament can affect its assembly dynamics. Today, experiments addressing this issue at the scale of individual actin filaments are emerging and bring novel insight into mechanotransduction. For instance, recent data show that actin filaments can act as mechanosensors, as an applied tension or curvature alters their conformation and their affinity for regulatory proteins. Filaments can also transmit mechanical tension to other proteins, which consequently change the way they interact with the filaments to regulate their assembly. These results provide evidence for mechanotransduction at the scale of individual filaments, showing that forces participate in the regulation of filament assembly and organization. They bring insight into the elementary events coupling mechanics and biochemistry in cells. The experiments presented here are linked to recent technical developments, and certainly announce the advent of more exciting results in the future.     

Contractile forces contribute to increased glycosylphosphatidylinositol-anchored receptor CD24-facilitated cancer cell invasion

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.     

A mathematical model for dorsal closure

During embryogenesis, drosophila embryos undergo epithelial folding and unfolding, which leads to a hole in the dorsal epidermis, transiently covered by an extraembryonic tissue called the amnioserosa. Dorsal closure (DC) consists of the migration of lateral epidermis towards the midline, covering the amnioserosa. It has been extensively studied since numerous physical mechanisms and signaling pathways present in DC are conserved in other morphogenetic events and wound healing in many other species (including vertebrates).We present here a simple mathematical model for DC that involves a reduced number of parameters directly linked to the intensity of the forces in the presence and which is applicable to a wide range of geometries of the leading edge (LE). This model is a natural generalization of the very interesting model proposed in Hutson et al. (2003). Being based on an ordinary differential equation (ODE) approach, the previous model had the advantage of being even simpler, but this restricted significantly the variety of geometries that could be considered and thus the number of modified dorsal closures that could be studied.A partial differential equation (PDE) approach, as the one developed here, allows considering much more general situations that show up in genetically or physically perturbed embryos and whose study will be essential for a proper understanding of the different components of the DC process. Even for native embryos, our model has the advantage of being applicable since an early stages of DC when there is no antero-posterior symmetry (approximately verified only in the late phases of DC).We validate our model in a native setting and also test it further in embryos where the zipping force is perturbed through the expression of spastin (a microtubule severing protein). We obtain variations of the force coefficients that are consistent with what was previously described for this setting.     

Cell-substratum and cell-cell adhesion forces and single-cell mechanical properties in mono- and multilayer assemblies from robotic fluidic force microscopy

The epithelium covers, protects, and actively regulates various formations and cavities of the human body. During embryonic development the assembly of the epithelium is crucial to the organoid formation, and the invasion of the epithelium is an essential step in cancer metastasis. Live cell mechanical properties and associated forces presumably play an important role in these biological processes. However, the direct measurement of cellular forces in a precise and high-throughput manner is still challenging. We studied the cellular adhesion maturation of epithelial Vero monolayers by measuring single-cell force-spectra with high-throughput fluidic force microscopy (robotic FluidFM). Vero cells were grown on gelatin-covered plates in different seeding concentrations, and cell detachment forces were recorded from the single-cell state, through clustered island formation, to their complete assembly into a sparse and then into a tight monolayer. A methodology was proposed to separate cell-substratum and cell-cell adhesion force and energy (work of adhesion) contributions based on the recorded force-distance curves. For comparison, cancerous HeLa cells were also measured in the same settings. During Vero monolayer formation, a significantly strengthening adhesive tendency was found, showing the development of cell-cell contacts. Interestingly, this type of step-by-step maturation was absent in HeLa cells. The attachment of cancerous HeLa cells to the assembled epithelial monolayers was also measured, proposing a new high-throughput method to investigate the biomechanics of cancer cell invasion. We found that HeLa cells adhere significantly stronger to the tight Vero monolayer than cells of the same origin. Moreover, the mechanical characteristics of Vero monolayers upon cancerous HeLa cell influence were recorded and analyzed. All these results provide insight into the qualitative assessment of cell-substratum and cell-cell mechanical contacts in mono- and multilayered assemblies and demonstrate the robustness and speed of the robotic FluidFM technology to reveal biomechanical properties of live cell assemblies with statistical significances.     

Driving forces for data exchange

The subgroup ‘Driving Forces for Data Exchange’ as part of the SETAC LCA Workgroup on Data Availability and Quality is finishing its final report with recommendations and guidelines to stimulate availability and exchange of LCI data. Activities in the past three years involved a literature review, interviews with LCI data publishers and stakeholder discussions. The final report will be part of a SETAC ‘Code of Life Cycle Inventory Practice’, dealing with LCI data availability and quality aspects in a broader sense.     

Studies on the mechanism of freezing damage to mouse liver. IV. Effects of ultrarapid freezing on structure and function of isolated mitochondria

Mouse liver mitochondria isolated in 0.25 m sucrose were subjected to progressively increasing cooling rates by quench-thaw from liquid nitrogen, isopentane at ?155 °C, and liquid propane at ?185 °C. Structural damage, assessed by electron microscopy and by quantitation of supernatant protein, increased progressively with the cooling rate. Oxidative phosphorylation (with succinate as substrate) was destroyed at all three cooling rates, while acceptorless respiration (succinoxidase) showed a progressive increase with cooling rate, suggesting uncoupling. The succinate cytochrome c reductase system showed no functional damage. Dimethyl sulfoxide, 10–20% by volume, markedly improved structural preservation of the mitochondria, but did not restore oxidative phosphorylation, and further increased the degree of uncoupling.Upon resuspending the mitochondria in 0.15 m KCl prior to quench-thaw, the succinate cytochrome c reductase system displayed an optimal recovery after isopentane quench-thaw, with a sharp decline at still higher cooling rates, as had been encountered in tissue slice experiments, suggesting a compartmental ice-transition in mitochondria over this range of cooling rates. Structurally, however, the KCl-resuspended mitochondria were equally and maximally disrupted by all three quench-thaw procedures. Sixty percent of the mitochondrial protein was extruded into the supernate, far above the levels released from sucrose-suspended mitochondria by quench-thaw and significantly above the 45% released by sonication. Compared to isotonic KCl, isotonic sucrose was thus providing full cryoprotection for the reductase complex and moderate protection for mitochondrial structure. The discrepancies among the several structural and functional indicators of mitochondrial damage leave little possibility that a single compartmental ice-transition, occurring over this range of cooling rates, could provide a coherent explanation for freezing damage to liver mitochondria.